Alpha 1,4-galactosyltransferase and dna encoding thereof

ABSTRACT

The object of the present invention is to provide α1,4-galactosyltransferase to transfer a galactose residue to C4 position of galactose residue of lactosylceramide or galactosylceramide, and DNA coding for the enzyme. 
     What is provided includes the following polypeptides (a) and (b), and DNAs encoding thereof:
         (a) a polypeptide consisting of an amino acid sequence represented by the amino acid Nos. 46-353 in SEQ ID NO: 2; or   (b) a polypeptide which comprises an amino acid sequence including substitution, deletion, insertion or transposition of one or few amino acids in the amino acid sequence of (a) and which has an enzymatic activity to transfer a galactose residue from a galactose donor to C4 position of galactose residue of lactosylceramide or galactosylceramide which serves as an acceptor.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the production of a polypeptide using arecombinant DNA, or to a tool useful for diagnosis or treatment ofdiseases, or more specifically to α1,4-galactosyltransferase and DNAencoding thereof, a recombination vector containing the DNA, and atransformed cell transfected with the DNA or by the recombinationvector, and to a method for producing Gb3/CD77 or globo-seriesglycolipids by using the transformed cell.

2. Description of the Related Art

Glycosphingolipids are amphipathic molecules(ref.1) that are synthesizedby sequential actions of glycosyltransferases(ref.2). Addition of one ofthree different sugars onto lactosylceramide (which may be termed“LacCer” hereinafter) results in the synthesis of either one of threemajor glycolipid series, i.e., ganglioside-series (a 2,3-sialic acid),lacto/neolacto-series (β1,4-N-acetylglucosamine) and globo-series(α1,4-galactose). Although a number of genes coding for enzymesresponsible for the synthesis of the carbohydrate moiety ofglycosphingolipids have been recently isolated(ref.3), noglycosyltransferase genes specific for the synthesis of globo-seriesglycolipids have been isolated to date.

Globotriaosylceramide (hereinafter sometimes referred to as “Gb3”) issynthesized by α1,4-galactosyltransferase (α1,4 Gal-T) fromLacCer(ref.4). This glycolipid has been characterized on red blood cellsas the P^(k) antigen of the P blood group system(ref.5). Since Wiels etal(ref.6) reported that Gb3 was a Burkitt's lymphoma associated antigen,the expression and biological significance of Gb3 have been vigorouslystudied(ref.7, 8 and 9). Since Gb3 was clustered as CD77, this antigenwill be referred to as Gb3/CD77.

Gb3/CD77 was reported to be expressed in high amounts on Burkitt'slymphoma cells. However, it is now considered to be a differentiationantigen expressed on B cells, and can also be found in some malignanttumors of B cell lineage(ref.7). Among normal leukocytes, it is onlyexpressed on a subset of tonsillar B cells in the germinal centers(GC)(ref.9). Interestingly, GC B lymphocytes expressing Gb3/CD77 undergorapid and spontaneous apoptosis when isolated and cultured invitro(ref.11). Furthermore, Burkitt's lymphoma cells with Gb3/CD77antigen were also easily induced to enter apoptosis upon culture at lowserum concentration or cross-linking by anti-immunoglobulin Mantibodies(ref.12).

Gb3/CD77 has been recognized as a receptor for verotoxins (VTs), theShiga-like toxin from E. coli 0157 strain that can trigger seriouscytotoxic effects(ref.13 and 14). VT B-subunit specifically binds toGb3/CD77, then A subunit is incorporated into cells, resulting in thedegradation of 28 S ribosomal RNA and cell death(ref.15). However, onlyB-subunit is also able to induce apoptosis when cross-linked(ref.16).These results indicate that Gb3/CD77 is a critical molecule in mediatingapoptosis signals, although the precise mechanisms remain to beinvestigated.

As stated above, it was revealed that Gb3/CD77, a globo-seriesglycolipid, is synthesized by addition of α1,4-galactose toLacCer(ref.4), but the glycosyltransferase specific for this synetheticreaction has not been isolated yet. An object of the present inventionis to isolate a Gb3/CD77 synthase, that is, a 1,4-galactosyltransferase,and DNA encoding thereof, and to provide the use thereof.

The present inventors have studied hard to achieve the above objects,and succeeded in isolating DNA encoding α1,4-galactosyltransferase,revealing its nucleotide sequence, and confirming that the DNA isresponsible for the expression of active α1,4-galactosyltransferase.Thus, they achieved the present invention.

SUMMARY OF THE INVENTION

The present invention provides a polypeptide of (a) or (b) below(hereinafter sometimes referred to as “the polypeptide of the presentinvention”):

(a) a polypeptide consisting of an amino acid sequence represented bythe amino acid Nos. 46-353 in SEQ ID NO: 2; or

(b) a polypeptide which comprises an amino acid sequence includingsubstitution, deletion, insertion or transposition of one or few aminoacids in the amino acid sequence of (a) and which has an enzymaticactivity to transfer a galactose residue from a galactose donor to C4position of galactose residue of lactosylceramide or galactosylceramidewhich serves as an acceptor.

The polypeptide of the present invention also includes a polypeptide of(a′) or (b′) below:

(a′) a polypeptide consisting of an amino acid sequence represented bythe amino acid Nos. 20-353 in SEQ ID NO:2; or

(b′) a polypeptide which comprises an amino acid sequence includingsubstitution, deletion, insertion or transposition of one or few aminoacids in the amino acid sequence of (a′) and which has an enzymaticactivity to transfer a galactose residue from a galactose donor to C4position of galactose residue of lactosylceramide or galactosylceramidewhich serves as an acceptor.

The polypeptide of the present invention also includes a polypeptide of(a″) or (b″) below:

(a″) a polypeptide consisting of an amino acid sequence represented bySEQ ID NO:2; or

(b″) a polypeptide which comprises an amino acid sequence includingsubstitution, deletion, insertion or transposition of one or few aminoacids in the amino acid sequence of (a″) and which has an enzymaticactivity to transfer a galactose residue from a galactose donor to C4position of galactose residue of lactosylceramide or galactosylceramidewhich serves as an acceptor.

The present invention also provides a DNA encoding the polypeptidesaccording to any one of above polypeptides (hereinafter sometimesreferred to as “the DNA of the present invention”). The DNA of thepresent invention includes a DNA represented by (a) or (b) below:

-   -   (a) a DNA comprising a nucleotide sequence represented by        nucleotide Nos. 269 to 1192 in SEQ ID NO:1; or    -   (b) a DNA hybridizable with a DNA comprising a nucleotide        sequence represented by SEQ ID NO:1, a nucleotide sequence        complimentary to SEQ ID NO:1, or a part of those sequences,        under a stringent condition.

The DNA of the present invention also includes a DNA encoding apolypeptide having an enzymatic activity to transfer a galactose residuefrom a galactose donor to C4 position of galactose residue oflactosylceramide or galactosylceramide which serves as an acceptor.

The present invention still further provides a recombination vectorcontaining the DNA of the present invention.

The present invention still further provides a transformed cell obtainedby transfecting a host cell with the DNA of the present invention or therecombination vector.

The present invention still further provides a method for producing thepolypeptide of the present invention, comprising the steps of:

producing the polypeptide of the present invention by culturing thetransformed cell above in a medium; and

recovering said polypeptide from the medium and/or a cell extract of thecultured transformed cell.

10. The present invention still further provides a method

for producing Gb/CD77 comprising the steps of: exposing the polypeptideaccording to any one of claims 1 to 3, or a cultured product of thetransformed cell according to claim 8, to lactosylceramide, to causethereby enzymatic reaction; and recovering Gb3/CD77.

The present invention still further provides a method for producing aglycolipid as represented by the following formula (I) comprising thesteps of:

exposing the polypeptide of the present invention, or a cultured productof the transformed cell above, to galactosylceramide, to cause therebyenzymatic reaction; and

recovering the glycolipid represented by the following formula (I):

Galα1→4Gal-Cer  (1)

wherein Gal represents a galactose residue, Cer represents a ceramideresidue and α1→4 represents an α1-4 glycosidic linkage.

In the present invention, the enzyme having an activity to transfer agalactose residue from a galactose donor to C4 position of galactoseresidue of lactosylceramide or galactosylceramide which serves as anacceptor, will be called “α1,4-galactosyltransferase.” Further, theactivity of the enzyme to transfer a galactose residue from a galactosedonor to C4 position of galactose residue of lactosylceramide orgalactosylceramide which serves as an acceptor, will be called“α1,4-galactosyltransferase activity.”

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows flow cytometry indicating the expression of Gb3/CD77 by Lcells. The left diagram relates to L cells transfected with pCDM8 whilethe right diagram to L cells transfected with pVTR1/CDM8. The thick lineindicates the result of cells stained with mAb38.13 and FITC-conjugatedrabbit anti-rat IgG (secondary antibodies) while the thin line theresult of cells stained only with the secondary antibodies (control).

FIG. 2 shows TLC charts of glycolipids extracted from cells transientlytransfected with α1,4 Gal-T gene.

A: TLC of glycolipids extracted from L cells transfected with pCDM8 (VC)or pVTR1/CDM8 (TF). RBC represents neutral glycolipids extracted fromhuman B red blood cells.

B: TLC immunostaining of Gb3/CD77 by mAb38.13.

FIG. 3 shows the hydropathy plot of a polypeptide of the presentinvention.

FIG. 4 shows the α1,4 Gal-T activity in the extracts of transienttransfectants of pVTR1.

A: α1,4 Gal-T activity when LacCer was used as an acceptor.

B: α1,4 Gal-T activity when various acceptors were used. PG representsparagloboside.

FIG. 5 shows the result of northern blotting of α1,4 Gal-T gene.

A: the upper columns show the results of hybridization with a³²P-labeled probe derived from pVTR1, while the lower columns show theresults of hybridization of the same membranes as in A with a β-actincDNA probe (control).

B: the expression levels of mRNA of α1,4 Gal-T gene were compared amongvarious human tissues. The ordinate represents the percentage of theexpression level of a given tissue with respect to the level of heartafter correction with the control.

FIG. 6 shows flowcytometry of stable transfectant cells. The leftdiagram relates to cells transfected with pSV2neo while the rightdiagram to cells transfected with pVTR1 and pSV2neo. The thin lineindicates the number of cells stained with mAb38.13 and FITC-conjugatedrabbit anti-rat IgG (secondary antibodies) while the thick line thenumber of cells stained only with the secondary antibodies (control).

FIG. 7 shows the results of MTT assay of L-neo and L-VTR1. The leftgraph shows the result of L-neo while the right one the result ofL-VTR1.

FIG. 8 shows the effect of vero toxins on the cell growth.

FIG. 9 shows an electrophoresis indicating the result of DNAfragmentation assay.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The mode for carrying out the present invention is described below.

<1> The Polypeptide of the Present Invention

The polypeptide of the present invention is a polypeptide of (a) or (b)below:

(a) a polypeptide consisting of an amino acid sequence represented bythe amino acid Nos. 46-353 in SEQ ID NO: 2; or

(b) a polypeptide which comprises an amino acid sequence includingsubstitution, deletion, insertion or transposition of one or few aminoacids in the amino acid sequence of (a) and which has an enzymaticactivity to transfer a galactose residue from a galactose donor to C4position of galactose residue of lactosylceramide or galactosylceramidewhich serves as an acceptor.

The above polypeptides contain at least a catalytic domain ofα1,4-galactosyltransferase as will be described later.α1,4-galactosyltransferase comprises, in order from its N-terminal, acytoplasmic domain, transmembrane domain, and catalytic domain. Thepolypeptides described above in (a′) and (b′) comprise at least thetransmembrane and catalytic domains. The polypeptides described above in(a″) and (b″) comprise the cytoplasmic, transmembrane and catalyticdomains. These peptides are all included in the polypeptides of thepresent invention.

An example of the amino acid sequence of a polypeptide of the presentinvention is represented in SEQ ID NO:2. In SEQ ID NO:2, amino acid Nos.1-19 represents the cytoplasmic domain, Nos. 20-45 the transmembranedomain, and Nos. 46-353 the catalytic domain.

Among those polypeptides, the polypeptides (a), (a′) and (a″) arepreferred; the polypeptides (a′) and (a″) are more preferred; and thepolypeptide (a″) is most preferred. However, any one of them may be usedas long as it has an α1,4-galactosyltransferase activity.

In this specification, the term “a polypeptide which comprises an aminoacid sequence including substitution, deletion, insertion ortransposition of one or few amino acids and which has an enzymaticactivity to transfer a galactose residue from a galactose donor to C4position of galactose residue of lactosylceramide or galactosylceramidewhich serves as an acceptor” means that, one or more amino acid residuesof the polypeptide may be substituted, deleted, inserted, or transferredas long as such modification does not substantially affect the abilityto the enzymatic activity (α1,4 Galactosyltransferase activity) of thepolypeptide to transfer a galactose residue from a galactose donor to C4position of galactose residue of lactosylceramide or galactosylceramidewhich serves as an acceptor.

Mutation such as substitution, deletion, insertion, or transposition ofamino acid residues may occur in the amino acid sequence of thepolypeptides existing in nature due to, for example, the modifyingreaction of the biosynthesized polypeptides in the living organisms orduring their purification as well as polymorphism and mutation of theDNAs encoding the polypeptides, nevertheless some of mutatedpolypeptides are known to have substantially the same physiological andbiological activities as the intact polypeptides that have not beenmutated. The polypeptide of the present invention includes those havingslightly different structures but not having a significant difference inthe functions. The polypeptide of the present invention also includesthose which have been artificially treated to have mutation as describedabove in the amino acid sequences. In this case, a further variety ofmutants can be produced. For example, a polypeptide having a humaninterleukin 2 (IL-2) amino acid sequence, in which a cysteine residuehas been replaced with a serine residue, is known to retain theinterleukin 2 activities (Science 224, 1431 (1984)). Furthermore, apolypeptide of certain kind is known to have a peptide region that isnot essential for exhibiting its activities. Examples of suchpolypeptides include a signal peptide contained in a polypeptide that issecreted extracellularly and a pro-sequence found in a precursor ofprotease, and the like. Most of these regions are removed aftertranslation or upon conversion into an active form of the polypeptides.These polypeptides exist in different primary structures but finallyhave equivalent functions. Such polypeptides are also included in thepolypeptide of the present invention.

The term “few amino acids” used herein means the number of amino acidresidues that may be mutated to the extent that the enzymatic activitiesof the polypeptide of the present invention are not lost. For example,in a polypeptide consisting of 400 amino acid residues, about 2 to 20,preferably 2 to 10, more preferably 2 to 5 or less of amino acidresidues may be mutated.

The α1,4-galactosyltransferase activity can be assayed by a knownmethod(ref.4). Specifically, the assay consists of using UDP-galactose(UDP-Gal) as a donor, and depending on the reaction where galactose istransferred by the enzyme to Laccer (acceptor). From above, it isobvious that any one skilled in the art could easily selectsubstitution, deletion, insertion or transposition of one or more aminoacid residues which does not substantially affect the enzymaticactivity, using its α1,4-galactosyltransferase activity as an index.

The polypeptide of the present invention was obtained as follows:

cDNA of α1,4-galactosyltransferase was isolated from human melanoma cellline; the cDNA was expressed in mouse fibroblasts; and the peptide wasidentified and characterized, and its structure was determined. Thepolypeptide of the present invention may be obtained by expressing theDNA of the present invention as described later, in appropriate cells.The same polypeptides chemically synthesized are naturally included inthe present invention. The method for producing the polypeptide of thepresent invention using the DNA of the present invention will bedescribed later.

The polypeptide of the present invention is not necessarily a singlepolypeptide but may be a part of a fusion protein if necessary. A fusionprotein comprising the polypeptide of the present invention and anotherpolypeptide such as protein A may be cited as such an example.

The polypeptide of the present invention may consist of a polypeptidealone, or contain a sugar chain or the like, as long as it has theenzymatic activity to transfer a galactose residue from a galactosedonor to C4 position of galactose residue of lactosylceramide orgalactosylceramide which serves as an acceptor.

<2> DNA of the Present Invention

The DNA of the present invention is a DNA encoding the polypeptide ofthe present invention as described above. The DNAs encoding thepolypeptides described below in (a) and (b) may be cited as an example:

(a) a polypeptide consisting of an amino acid sequence represented bythe amino acid Nos. 46-353 in SEQ ID NO: 2; or

(b) a polypeptide which comprises an amino acid sequence includingsubstitution, deletion, insertion or transposition of one or few aminoacids in the amino acid sequence of (a) and which has an enzymaticactivity to transfer a galactose residue from a galactose donor to C4position of galactose residue of lactosylceramide or galactosylceramidewhich serves as an acceptor.

Among the DNAs, the one coding for polypeptide (a) is more preferred.

The DNA encodes at least the catalytic domain ofα1,4-galactosyltransferase, but the DNA of the present invention alsoincludes DNAs encoding, in addition to above, the polypeptides includinga transmembrane domain and/or cytoplasmic domain.

The DNA encoding the polypeptide (a) includes, for example, a DNAcontaining a nucleotide sequence represented by nucleotide Nos. 269-1192in SEQ ID NO:1. The DNA encoding the polypeptide containing thetransmembrane domain includes, for example, a DNA containing anucleotide sequence represented by nucleotide Nos. 191-1192 in SEQ IDNO:1. The DNA encoding the polypeptide containing the cytoplasmic domainincludes, for example, a DNA containing a nucleotide sequencerepresented by nucleotide Nos. 134-1192 in SEQ ID NO:1.

Furthermore the DNA comprising a nucleotide sequence represented by SEQID NO:1 has been derived from human originally. As a matter of course,however, the DNA of the present invention is not limited to any source,and includes those that are produced by genetic engineering procedure orchemical synthesis.

Furthermore, any one ordinarily skilled in the art would readilyunderstand that the DNA of the present invention includes DNAs havingnucleotide sequences different from what is described above due todegeneracy of the genetic codes.

The DNA of the present invention also includes DNA or RNA complementaryto the DNA of the present invention. Furthermore, the DNA of the presentinvention may be either a single-stranded coding chain encoding thepolypeptide of the present invention or a double-stranded chainconsisting of the above single-stranded chain and a DNA or an RNA havinga complementary nucleotide sequence thereto.

The DNA of the present invention was obtained by expression cloning aswill be described later. However, since the nucleotide sequence of theDNA of the present invention was determined, it will be possible toisolate the same DNA from human-derived mRNA or cDNA, or a chromosomalDNA through PCR with an oligonucleotide prepared from the nucleotidesequence thus determined to serve as a primer, or from a cDNA library orchromosomal DNA library through hybridization with an oligonucleotideprepared from the nucleotide sequence thus determined to serve as aprobe.

The gene encoding the polypeptide of the present invention derived froma chromosome is expected to contain introns in the coding region. DNAfragments separated by introns are also included in the DNA of thepresent invention.

The DNA of the present invention may include DNAs, as long as they codefor the polypeptides having an enzymatic activity to transfer agalactose residue from a galactose donor to C4 position of galactoseresidue of lactosylceramide or galactosylceramide which serves as anacceptor, hybridizable with a probe comprising a nucleotide sequencecomplimentary to the nucleotide sequence of SEQ. ID No:1, or to anucleotide sequence represented by nucleotide Nos. 269-1292, nucleotidesequence represented by nucleotide Nos. 191-1292, or nucleotide sequencerepresented by nucleotide Nos. 134-1292 of SEQ ID No:1, or with a probecomprising a part of those nucleotide sequences, under a stringentcondition. The “stringent condition” here refers to a condition underwhich a so-called specific hybrid is formed, but no non-specific hybridsare formed (see Sambrook, J. et al., Molecular Cloning A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press (1989)). The“stringent condition” may include for example subjecting a test DNA to asolution containing 50% formamide, 4×SSC, 50 mM HEPES (pH 7.0),10×Denhardt's solution, and 100 μg/ml of sermon sperm DNA, allowing itto hybridize in the solution at 42° C., and washing the yield in 2×SSCand 0.1% SDS solution at room temperature, then in 0.1×SSC and 0.1% SDSsolution at a temperature of 50° C. or less.

Production of the polypeptide of the present invention may be achievedby cultivating transformed cells transfected with the DNA of the presentinvention on appropriate growth medium, thereby allowing the polypeptideof the present invention encoded by the DNA of the present invention toexpress itself, and by recovering the polypeptide thus expressed. Thethus expressed polypeptide of the present invention can be extractedfrom a cultured product of the transformed cells (comprising bothtransformed cells and medium). However, if the polypeptide of thepresent invention is accumulated in the cytoplasm of transformed cells,or their membrane fraction, the polypeptide must be extracted from thetransformed cells. Or, if the polypeptide is accumulated in medium, itmust be extracted from medium. Or, if use of the transformed cells inwhich the polypeptide is expressed is desired, the transformed cellsthemselves, or their processed products may be used intact, or afterthey have been bound to an appropriate solid phase, or covered with gelfor solidification. The “transformed cell” includes not only transformedcell themselves but also extracts from them.

For the transfection of the DNA of the present invention into a hostcell, it is only necessary to prepare a recombination vector byinserting the DNA of the present invention into an appropriate vector,and to introduce the DNA of the present invention into a host cellthrough the recombination vector. The vector is preferably an expressionvector.

The host cell is not limited to any specific cells, as long as they canfully play the function of the DNA of the present invention, or of therecombination vector containing the DNA of the present invention. Thus,it may include any animal cells, plant cells, micro-organisms(bacteria), or the like. Procaryotic cells such as E. coli, oreucaryotic cells such as mammalian cells may be exemplified. When aprocaryotic cell such as E. coli is used, addition of sugar chain doesnot occur to the polypeptide produced as by expression of the DNA of thepresent invention, then the polypeptide of the present invention havingno sugar chain can be obtained. When eucaryotic cell such as a mammaliancell is used, sugar chain may add to the polypeptide produced byexpression of the DNA of the present invention, then the form of thepolypeptide of the present invention comprising sugar chain can beobtained.

Specifically, the host cell to transfect with the DNA of the presentinvention may include for example L cells derived from mousefibroblasts. Specifically, the vector may include pCDM8 or pcDNA3expression vector (both available from Invitrogen). The culture mediumand condition may be chosen appropriately according to a given hostcell.

The DNA of the present invention may be expressed directly.Alternatively, it may be expressed with another polypeptide as a fusionpolypeptide. The full-length DNA of the present invention may beexpressed. It may also be expressed in part as a partial peptide.

The method for introducing the DNA of the present invention may dependon transfection based for example on DEAE-dextran method.

Recovering the polypeptide of the present invention from a culturedproduct may be performed by known extraction and purification methodsfor polypeptides. The cultured product used herein includes the mediumand the cells in the medium.

Extraction of the polypeptide of the present invention may be performed,for example, by a method using a nitrogen cavitation apparatus,extraction from the cells disrupted by homogenization, glass beadmilling, sonic wave treatment, osmotic shock, freeze-thawing procedureor the like, extraction by using detergent, or combination of thosemethods.

When the DNA of the present invention encoding the polypeptide (a″) isexpressed in L cells, the polypeptide of the present invention islocalized at the membrane fraction of the cell. When the DNA of thepresent invention is expressed as a fusion protein comprising apolypeptide of the present invention (or a part thereof), and anotherpeptide, so as to be a soluble protein, that fusion protein may bepresent in the cytoplasm. When the DNA of the present invention isexpressed as a fusion protein comprising the polypeptide of the presentinvention or a part thereof and a secretion signal, the resultingprotein may be secreted into medium. Isolation of DNA encoding a part ofthe polypeptide of the present invention may be achieved by preparing aprimer previously designed to produce such a DNA, and applying theprimer in PCR to human derived mRNA, cDNA library, or chromosomal DNA.

Specific examples of the method of purifying the polypeptide of thepresent invention extracted from the cells or medium include salting outwith salt such as ammonium sulfate or sodium sulfate, centrifugation,dialysis, ultrafiltration, absorption chromatography, ion exchangechromatography, hydrophobic chromatography, reverse phasechromatography, gel filtration, gel permeation chromatography, affinitychromatography, electrophoresis, and any combination thereof.

It can be confirmed whether the polypeptide of the present invention hasbeen produced or not by analyzing amino acid sequence, action, andsubstrate specificity of the purified polypeptide.

<3> Utilization of the Polypeptide and DNA of the Present Invention

The polypeptide of the present invention can be utilized for thesynthesis of globo-series glycolipids, for example, for the synthesis ofGb3/CD77 by exposing the polypeptide of the present invention or acultured product of the transformed cells transfected with the DNA ofthe present invention to lactosylceramide, thereby evoking enzymaticreaction. Also, it is possible to obtain a glycolipid as represented bythe following formula (I) by exposing the polypeptide of the presentinvention or a cultured product of the transformed cells transfectedwith the DNA of the present invention to galactosylceramide.

Gal α1→4Gal-Cer  (1)

In this procedure, exposure to substrate may occur through contact withthe transformed cells, if the polypeptide of the present invention isproduced and accumulated in the cytoplasm or in the membrane fraction,or through contact with medium if the polypeptide is accumulated inmedium. When the cells in which the polypeptide of the present inventionhas been expressed are utilized, exposure to substrate may occur throughdirect contact with the cells themselves, or extracts therefrom, orimmobilized extracts. The term “transformed cell” here includes not onlytransformed cells themselves but also extracts from them.

The polypeptide of the present invention is capable of specificallyattaching a galactose residue to C4 position of galactose residue oflactosylceramide or galactosylceramide contained in a sugar chain of aglycoprotein. Further, the polypeptide of the present invention isutilized for selective synthesis of a sugar chain.

Although a number of members of β1,3-galactosyltransferases (β1,3Gal-Ts)or β1,4Gal-Ts have been identified(ref.27-30), this gene is the firstand only α1,4Gal-T gene isolated so far. Moreover, no homologous genesto this gene were detected in the data base of C. elegans or Drosophilamelanogaster genes, even though many β1,4 and β1,3Gal-T-related geneshave been identified. These facts may indicate that α1,4Gal-T geneevolved relatively later than other galactosyltransferase genes, andglobo-series glycolipids synthesized through Gb3 are playing moreprecise roles compared to glycolipids of the other series.

Gb3/CD77 seems to be unusual because it can mediate various apoptoticsignals in both normal cells and malignant tumor cells, even though itdoes not contain any cytoplasmic domain(ref.16 and 31). The observedrapid death of CD77⁺ BC B cells in vitro suggests that endogenous ligandmolecules interact with Gb3/CD77 to bring about the physiologicselection of immature B cells(ref.11 and 32). Furthermore, thecapability of B subunit of VT to induce apoptosis of Gb3/CD77⁺cells(ref.16) strongly encourages the investigation ofGb3/CD77-associating cytoplasmic molecules(ref.31). Investigations ofthese ligands and signal transducers relevant to Gb3/CD77 mightcontribute to further understanding of the B cell selection and of thepathogenesis of hemolytic uremic syndrome caused by E. coli 0157infection. In particular, the tissue specificity of the syndrome such asrenal failure, hemolysis and neurological disorders, might be clarifiedby gene manipulation of the cloned Gb3/CD77 synthase.

Furthermore, it has recently been reported that Gb3/CD77 and gangliosideGM3 may function as alternative cofactors for the entry of humanimmunodeficiency virus type 1 (HIV-1) in CD4-induced interactionsbetween gp120 and glycosphingolipid microdomains(ref.33 and 34). If thisis the case, Gb3/CD77 may be a receptor not only for bacterial toxinsbut for viruses, and the regulation of Gb3/CD77 expression could be akey target for the therapeutic approaches of viral infections such asHIV-1.

Further, the expression of Gb3/CD77 in the kidney has been thought to berelated with the development of hemolytic uremic syndrome (HUS).Furthermore, Fabry's disease is known as a disease in which Gb3/CD77accumulates in the kidney, heart, brain and vasculature. From above, thepolypeptide and DNA of the present invention, and the method of thepresent invention for producing Gb3/CD77 or a glycolipid may serve as atherapeutic tool, diagnostic tool or research tool for the treatment ofdiseases caused by the abnormal expression of Gb3/CD77 as describedabove.

EXAMPLE

The present invention will be described more in detail below by means ofexamples.

Example 1 Isolation of cDNA for α1,4-Galactosyltransferase (α1,4 Gal-T)

<1> Preparation of a cDNA Library from a Human Melanoma Cell Strain, andCloning of α1,4 Gal-T cDNA.

A cDNA was prepared from poly(A⁺) RNA of a human melanoma cell lineSK-MEL-37 as described(ref.17). The cDNA library was constructed byinserting the cDNA into a vector plasmid pCDM8 (Invitrogen). The librarycontained 5×10⁶ independent colonies. The strain of bacteria was E. coliMC1061/P3 (ref.18).

Since the SK-MEL-37 cell line does not express Gb3/CD77 on its surface,it highly efficiently expresses α1,4 Gal-T, and thus the cDNA libraryprepared from this cell line is excellent for the present purpose.

Plasmids of the cDNA library were transfected into a mouse fibroblast Lcells together with pd13027 (polyoma T gene, provided by Dr. C. Basilicoat New York University, New York) using DEAE-dextran as described(ref.18). L cells express a large amount of LacCer although they have noα1,4 Gal-T activity nor Gb3/CD77 expression(ref.19). The L cells,because of these characteristics, served as an excellent host in thecloning of cDNA for α1,4 Gal-T. The L cell was kindly provided by Dr. A.P. Albino at Sloan-Kettering Cancer Center, New York, and was maintainedin Dulbecco's modified Eagle's minimal essential medium (DMEM)containing 7.5% of fetal bovine serum (FCS).

After 48 h, the transfected cells were detached and incubated with a ratmonoclonal antibody (mAb) 38.13 (ref.6) on ice for 45 min. Afterwashing, cells were plated on dishes coated with rabbit anti-rat IgM(ZYMED) as described (ref.17). Plasmid DNA was rescued from the pannedcells by preparing Hirt extracts, and transformed into MC1061/P3. Thesame procedure was repeated 5 times. The plasmid DNA was collected fromthe transformed cells.

Using microscale transfection of L cell and immunofluorescence assay,cDNA clones that determined the Gb3/CD77 expression were isolated. Cellsurface expression of Gb3/CD77 was analyzed by flow cytometry (BectonDickinson) as described (ref.19). MAbs 38.13 or TU-1 (23) were used withFITC-conjugated rabbit anti-rat IgG or anti-mouse IgM (ZYMED),respectively. As a result, two clones showing positive reactions weresuccessfully isolated. As described later, the two clones areessentially similar in nature, and thus one of them is called pVTR1, andfurther analysis was performed on that one.

FIG. 1 shows the results of flow cytometry of L cells transfected withthe plasmid pVTR1 or the plasmid pCDM8 (containing no target sequence toserve as control). It is obvious from this that the cells transfectedwith pVTR1 express Gb3/CD77 while those transfected with pCDM8 alone donot express Gb3/CD77. It was thus demonstrated that α1,4 Gal-T cDNAinserted to pVTR1 is involved in the synthesis of Gb3/CD77.

<2> Extraction of Glycolipids from the Transformed Cells, andIdentification of Gb3/CD77

Glycolipids were extracted as described(ref.21). Briefly, glycolipidswere extracted from about 400 μl of packed cells usingchloroform/methanol (2:1, 1:1, 1:2) sequentially. TLC was performed on ahigh performance TLC plates (MERCK, Darmstadt) using the solvent systemchloroform:methanol:0.22% CaCl₂ (60:35:8) and sprayed by orcinol. Forstandards, bovine brain ganglioside mixture (Wako, Tokyo), neutralglycolipids from human erythrocytes, and Gb3 (Sigma) were used.

Glycosphingolipids extracted from the transformed cells showed definiteGb3 bands in TLC, although the transformed cells with pCDM8 alone showedno Gb3 band (FIG. 2A), suggesting that the cloned pVTR1 derived fromα1,4Gal-T gene.

The identity of Gb3/CD77 was confirmed by TLC-immunostaining using analuminum-backed silica plate (MERCK) as described(ref.21). After TLC,the plate was blotted onto PVDF membrane as described(ref.22). Afterblocking, the plate was incubated with mAb, then antibody binding wasdetected with ABC kit (Vector Laboratories, Burlingame, Calif.) andKonica Immunostaining HRP-1000 (Konica, Tokyo). This TLC-immunostainingrevealed strong bands of Gb3 only in the extracts from the cDNAtransfected cells (FIG. 2B).

<3> Nucleotide Sequencing of Gene for α1,4 Gal-T

The nucleotide sequence of the cDNA clone which was confirmed to expressGb3/CD77 as described above was determined by dideoxynucleotidetermination sequencing using the PRISM dye terminator cycle sequencingkit and model 310 DNA sequencer (Applied Biosystems). The sequencingshowed that the two clones are essentially the same in sequence.Accordingly, one of them was selected for subsequent analysis, and namedpVTR1. The nucleotide sequence of cDNA in pVTR1 is shown by SEQ ID NO:1.The amino acid sequence encoded by this nucleotide sequence is shown bySEQ ID NOs:1 and 2.

The initiation codon is embedded within a sequence similar to the Kozakconsensus initiation sequence(ref. 24 and 25). This open reading framepredicts a 353-amino acid protein with a molecular mass of 40,498daltons.

Nucleotide and amino acid sequence homology search was carried out usingthe internet program BLAST (National Center for BiotechnologyInformation). However, no cDNA or protein having a high homology withthese sequences was found in the database.

Amino acid sequence and hydropathy analyses (35) were performed with asoftware GENETYX-MAC version 8.0 (Software Development, Tokyo)(FIG. 3).A single hydrophobic segment with 26 amino acids was present near theamino terminus (amino acid Nos. 20-45 in SEQ ID NO: 2). This putativesignal anchor sequence would place 19 residues within the cytoplasm and308 amino acids within the Golgi lumen.

The presence of two potential N-glycosylation sites are indicated (aminoacid sequence Nos. 121-123 and 203-205 in SEQ ID NO: 2). Relatively highfrequency of proline (10/31) was detected at the C′-side of thetransmembrane domain.

Example 2 Characterization and Production of α1,4 Gal-T <1> Enzyme Assayof α1,4 Gal-T

Membrane fractions were prepared as described(ref.19) from L cellstransfected with the gene for α1,4 Gal-T as obtained in Example 1. Theenzyme activity of α1,4 Gal-T in the membrane fraction was measured asdescribed previously(ref.4). The reaction mixture for the assaycontained the following in a volume of 50 μl: 50 mM sodiumcacodylate-HCl (pH 6.0), 10 mM MgCl₂, 5 mM galactonolactone (Sigma),0.3% Triton X-100 (Sigma), 0.4 mM (LacCer), 2.9 mM phosphatidylglycerol(Sigma), 0.2 mM UDP-Gal (Sigma), UDP-[¹⁴C] Gal (2.5×10⁵ dpm) (NEN), andmembrane fraction containing 50 μg protein. The protein concentrationwas determined by Lowry's methods(ref.20). The products was isolated bya C₁₈ Sep-Pak cartridge (Waters, Milford, Mass.) and analyzed by thinlayer chromatography (TLC) and autoradiography using a Bio-ImagingAnalyzer BAS2000 (Fuji Film, Tokyo). The results are shown in FIG. 4A.

L cells transfected with pVTR1/CDM8 showed high Gb3 synthase activity(7,012 units, pmol/h/mg of protein) when LacCer was used as an acceptor.On the other hand, L cells transfected with pCDM8 alone were completelynegative. Thus, this cDNA determined α1,4Gal-T activity and the surfaceexpression of Gb3/CD77, indicating that this cDNA encodes the Gb3/CD77synthase.

Enzyme activity toward other potential acceptors was also examined (FIG.4B). None of the acceptors examined except LacCer and galactosylceramideshowed significant levels of [¹⁴C]galactose incorporation (FIG. 4B). Kmvalues for these two substrates were 54.5 μM (LacCer) and 132 μM(galactosylceramide). The P1 antigen in the P blood group system is alsoformed by α1,4 galactose transfer (acting on paragloboside(PG)), but itwas confirmed that this enzyme is not responsible for the synthesis ofP1 antigen (FIG. 4B).

Example 3 Expression Analysis of α1,4 Gal-T Gene in Various Tissues(Northern Blotting)

Multiple Choice Northern Blots membranes (OriGene Technologies,Rockville, Mass.) were used. They were hybridized with [³²P]dCTP-labeledcDNA probe of pVTR1 or control β-actin as described (ref.18 and 19). Therelative expression levels of mRNA of α1,4Gal-T gene among human tissuesmeasured by Bio-Imaging Analyzer BAS2000 (Fuji Film) are presented as apercentage of the value of heart after correction with the control.Expression levels of the al, 4Gal-T gene in various human tissues wereexamined by Northern blotting. Among tissues examined, heart, kidney,spleen, liver, testis and placenta strongly expressed the gene (FIG. 5).

Example 4 Stable Transfection of Cells with pVTR1 Plasmid

To prepare stable transformants, pVTR1 and pSv2neo were co-transfectedinto L cells using Lipofection kit (TOYOBO, Tokyo, Japan). To selecttransformants, the cells were cultured in DMEM containing FCS (7.5%) andG418 (300 μg/ml). G481 is inactivated by 3′-O-aminoglycosidephosphotransferase encoded by the neo gene.

G418-resistant cells were cloned by limiting dilution. Clonestransfected with pSv2neo alone were prepared for control. These cellswere incubated together with mAb38.13, followed by addition ofFITC-conjugated rabbit anti-rat IgG for reaction, and the resultingcells were subjected to flow cytometry in the same manner as describedabove. The results showed that the cells transfected with pVTR1 andpSV2neo(L-VTR1) strongly expressed Gb3/CD77 while those transfected withpSV2neo alone(L-neo) did not express Gb3/CD77 (FIG. 6).

Example 5 Reaction of Transformed Cells to Verotoxins <1> MTT Assay

To compare the reactions of L-VTR1 and L-neo to VTs, MTT assay wasperformed using cells prepared in 48 well plates (1×10⁴ cells/well) andcultured in the presence of VT1 or VT2. The assay was performed bytriplicated samples. To quantify the cell proliferation, 50 μl of 5mg/ml of MTT (Sigma) in PBS was added to each well.

After incubation for 5 h at 37° C., the supernatants were aspirated and100 μl of n-propylalchohol containing 0.1% NP40 and 4 mM HCl was added.The color reaction was quantitated using automatic plate readerIMMUNO-MINI NJ-2300 (Nihon InterMed, Tokyo, Japan) at 590 nm with areference filter of 620 nm.

L-VTR1 in VT (+) medium showed marked growth suppression compared tothat cultured in the absence of VT, while L-neo showed no effects of VT(FIG. 7). MTT assay of L-VTR1 and L-neo after the exposure to variousconcentrations of VTs revealed marked growth suppression of L-VTR1 evenat 0.01 ng/ml, but not of L-neo (FIG. 8).

<2> DNA Fragmentation Assay

DNA fragmentation assay was performed to determine the mechanismresponsible for the death of L-VTR1 treated with VTs. Cells werecultured in the presence of VT2 (200 ng/ml). After 24 h, cells werecollected and the pellets were lysed in 100 μl of lysis buffer (10 mMTris-HCl pH 7.4, 10 mM EDTA and 0.5% Triton X-100) for 10 min at 4° C.After centrifugation, the supernatants were collected, and 2 μl of RNAse(10 mg/ml) and 2 μl of Proteinase K (10 mg/ml) were added. Afterincubation for 1 h at 37° C., the fragmented DNA was 2-propanolprecipitated. Electrophoresis was conducted using DNA derived from1.5×10⁶ cells in 2% agarose gel containing 0.2 μg/ml ethidium bromide inTEA buffer.

Agarose gel electrophoresis of cytoplasmic DNA extracted from L-VTR1revealed a clear pattern of DNA fragmentation characteristic ofapoptosis (FIG. 9). In contrast, the L-neo sample did not show anyladder formation. Thus, it was confirmed that Gb3/CD77 generated by thecDNA serves as a functional receptor for VTs.

The present invention provides α1,4-galactosyltransferase, and DNAencoding thereof. That enzyme can be utilized for the production ofglobo-series glycolipids such as Gb3/CD77.

Further, the DNA is useful for production of the above described enzyme,or serves as a therapeutic tool, diagnostic tool or research tool forthe treatment of diseases caused by the abnormal expression of Gb3/CD77,or it may be useful for the treatment or diagnosis of diseases involvedin the action of verotoxins.

REFERENCES

-   1. Wiegandt, H. (ed)(1985) in Glycolipids, pp. 199-260, Elsevier    Science Publishing Co., Inc., New York-   2. Paulson, J. C., and Colley, K. J. (1989) J. Biol. Chem. 264,    17615-17618-   3. Lloyd, K. O., and Furukawa, K. (1998) Glycoconj. J. 15, 627-636-   4. Taga, S., Mangeney, M., Tursz, T., and Wiels, J. (1995) Int. J.    Cancer 61, 261-267-   5. Marcus, D. M., Kundu, S. K., and Suzuki, A. (1981) Seminars in    Haematology, 18, 63-71-   6. Wiels, J., Fellous, M., and Tursz, T. (1981) Proc. Natl. Acad.    Sci. USA 78, 6485-6488-   7. Klein, G., Manneborg-Sandlund, A., Ehlin-Henriksson, B., Godal,    T., Wiels, J., and Tursz, T. (1983) Int. J. Cancer 31, 535-542-   8. Balana, A., Wiels, J., Tetaud, C., Mishal, Z., Tursz, T. (1985)    Int. J. Cancer 36, 453-460-   9. Murray, L. J., Habeshaw, J. A., Wiels, J., and    Greaves, M. F. (1985) Int. J. Cancer 36, 453-460-   10. Knapp, W. et al. (Eds) (1989) in Leukocyte typing IV, p. 118,    Oxford University Press-   11. Mangeney, M., Richard, Y., Coulaud, D., Tursz, T., and    Wiels, J. (1991) Eur. J. Immunol. 21, 1131-1140-   12. Gregory, C. D., Dive, C., Henderson, S., Smith, C. A.,    Williams, G. T., Gordon, J., and Rickinson, A. B. (1991) Nature 349,    612-614-   13. O'Brien, A. D., Lively, T. A., Chen, M. E., Rothman, S. W., and    Formal, S. B. (1983) Lancet, 1, 702-   14. Lingwood, C. A., Law, H., Richardson, S., Petric, M.,    Brunton, J. L., De Grandis, S., and Karmali, M. (1987) J. Biol.    Chem. 262, 8834-8839-   15. Endo, Y., Tsurugi, K., Yutsudo, T., Takeda, Y., Ogasawara, T.,    and Igarashi, K. (1988) Eur. J. Biochem. 171, 45-50.-   16. Mangeney, M., Lingwood, C. A., Taga, S., Caillou, B., Tursz, T.,    and Wiels, J. (1993) Cancer Res. 53, 5314-5319-   17. Seed B, Aruffo A (1987) Proc. Natl. Acad. Sci. USA 84, 3365-3369-   18. Nagata, Y., Yamashiro, S., Yodoi, J., Lloyd, K. O., Shiku, H.,    and Furukawa, K. (1992) J. Biol. Chem. 267, 12082-12089-   19. Yamashiro, S., Haraguchi, M., Furukawa, K., Takamiya, K.,    Yamamoto, A., Nagata, Y., Lloyd, K. O., Shiku, H., and    Furukawa, K. (1995) J. Biol. Chem. 270, 6149-6155-   20. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and    Randall, R. J. (1951) J. Biol. Chem. 193, 265-275-   21. Furukawa, K., Clausen, H., Hakomori, S., Sakamoto, J., Look, K.,    Lundblad, A., Mattes, M. J., and Lloyd, K. O. (1985) Biochemistry    24, 7820-7826-   22. Taki, T., Handa, S., and Ishikawa, D. (1994) Anal. Biochem. 221,    312-316-   23. Miyamoto, D., Ueno, T., Takashima, S., Ohta, K., Miyawaki, T.,    Suzuki, T., and Suzuki, Y. (1997) Glycoconj. J. 14, 379-388-   24. Kozak, M. (1986) Cell 44, 283-292-   25. Kozak, M. (1989) J. Cell Biol. 108, 229-241-   26. Wiels, J., Holmes, E. H., Cochran, N., Tursz, T., and    Hakomori, S. (1984) J. Biol. Chem. 259, 14783-14787-   27. Amado, M., Almeida, R., Carneiro, F., Levery, S. B., Holmes, E.    H., Nomoto, M., Hollingsworth, M. A., Hassan, H., Schwientek, T.,    Nielsen, P. A., Bennett, E. P., and Clausen, H. (1998) J. Biol.    Chem. 273, 12770-12778-   28. Schwientek, T., Almeida, R., Levery, S. B., Holmes, E. H.,    Bennett E, and Clausen, H. (1998) J Biol. Chem. 273, 29331-29340.-   29. Almeida, R., Amado, M., David, L., Levery, S. B., Holmes, E. H.,    Merkx, G., van Kessel, A. G., Rygaard, E., Hassan, H., Bennett, E.,    and Clausen, H. (1997) J. Biol. Chem. 272,-   30. Lo, N. W., Shaper, J. H., Pevsner, J., and Shaper, N. L. (1998)    Glycobiology 8, 517-526-   31. Taga, S., Carlier, K., Mishal, Z., Capoulade, C., Mangeney, M.,    Lecluse, Y., Coulaud, D., Tetaud, C., Pritchard, L. L., Tursz, T.,    and Wiels, J. (1997) Blood 90, 2757-2767-   32. Mangeney, M., Rousselet, G., Taga, S., Tursz, T., and    Wiels, J. (1995) Mol. Immunol. 32, 333-339-   33. Puri, A., Hug, P., Jernigan, K., Barchi, J., Kim, H.-Y.,    Hamilton, J., Wiels, J., Murray, G. J., Brady, R. O., and    Blumenthal, R. (1998) Proc. Natl. Acad. Sci. USA 95, 14435-14440-   34. Hammache, D., Yahi, N., Maresca, M., Pieroni, G., and    Fantini, J. (1999) J. Virol. 73, 5244-5248-   35. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132-   36. Svennerholm, L. (1963) J. Neurochem. 10, 613-623

1. A polypeptide of (a) or (b) below: (a) a polypeptide consisting of anamino acid sequence represented by the amino acid Nos. 46-353 in SEQ IDNO: 2; or (b) a polypeptide which comprises an amino acid sequenceincluding substitution, deletion, insertion or transposition of one orfew amino acids in the amino acid sequence of (a) and which has anenzymatic activity to transfer a galactose residue from a galactosedonor to C4 position of galactose residue of lactosylceramide orgalactosylceramide which serves as an acceptor.
 2. A polypeptide of (a′)or (b′) below: (a′) a polypeptide consisting of an amino acid sequencerepresented by the amino acid Nos. 20-353 in SEQ ID NO:2; or (b′) apolypeptide which comprises an amino acid sequence includingsubstitution, deletion, insertion or transposition of one or few aminoacids in the amino acid sequence of (a′) and which has an enzymaticactivity to transfer a galactose residue from a galactose donor to C4position of galactose residue of lactosylceramide or galactosylceramidewhich serves as an acceptor.
 3. A polypeptide of (a″) or (b″) below:(a″) a polypeptide consisting of an amino acid sequence represented bySEQ ID NO:2; or (b″) a polypeptide which comprises an amino acidsequence including substitution, deletion, insertion or transposition ofone or few amino acids in the amino acid sequence of (a″) and which hasan enzymatic activity to transfer a galactose residue from a galactosedonor to C4 position of galactose residue of lactosylceramide orgalactosylceramide which serves as an acceptor.
 4. A DNA encoding thepolypeptide according to any one of claims 1 to
 3. 5. The DNA accordingto claim 4 represented by (a) or (b) below: (a) a DNA comprising anucleotide sequence represented by nucleotide Nos. 269 to 1192 in SEQ IDNO:1; or (b) a DNA hybridizable with a DNA comprising a nucleotidesequence represented by SEQ ID NO:1, a nucleotide sequence complimentaryto SEQ ID NO:1, or a part of those sequences, under a stringentcondition.
 6. The DNA according to claim 5 encoding a polypeptide havingan enzymatic activity to transfer a galactose residue from a galactosedonor to C4 position of galactose residue of lactosylceramide orgalactosylceramide which serves as an acceptor.
 7. A recombinationvector containing the DNA as described in any one of claims 4 to
 6. 8. Atransformed cell obtained by transfecting a host cell with the DNAaccording to any one of claims 4 to 6, or the recombination vectoraccording to claim
 7. 9. A method for producing the polypeptideaccording to any one of claims 1 to 3, comprising the steps of:expressing the polypeptide according to claims 1 to 3 by culturing thetransformed cell according to claim 8 in a medium suitable forexpressing the polypeptide; and recovering said polypeptide from themedium and/or a cell extract of the cultured transformed cell.
 10. Amethod for producing Gb3/CD77, comprising the steps of: exposing thepolypeptide according to any one of claims 1 to 3, or a cultured productof the transformed cell according to claim 8, to lactosylceramide, tocause thereby enzymatic reaction; and recovering Gb3/CD77.
 11. A methodfor producing a glycolipid represented by the following formula (I),comprising the steps of: exposing the polypeptide according to any oneof claims 1 to 3, or a cultured product of the transformed cellaccording to claim 8, to galactosylceramide, to cause thereby enzymaticreaction; and recovering the glycolipid represented by the followingformula (I):Galα1→4Gal-Cer  (1) wherein Gal represents a galactose residue, Cerrepresents a ceramide residue and α1→4 represents an α1-4 glycosidiclinkage.